A. Integrins and Disintegrins
Integrins are a family of cell surface proteins that mediate adhesion between cells (cell-cell adhesion) and between cells and extracellular matrix proteins (cell-ECM adhesion). Integrins are heterodimeric structures composed of noncovalently bound α and β subunits. In humans, at least fifteen different α subunits and eight different β subunits combine to form integrins with diverse biological activities and ligand specificities. Integrins play important roles in biological processes including embryonic development, platelet aggregation, immune reactions, tissue repair and remodeling, bone resorption, and tumor invasion and metastasis. Integrins are, therefore, important targets for therapeutic intervention in human disease.
The disintegrins are a family of low molecular weight, soluble, cysteine-rich peptides which have been isolated from snake venom (reviewed in Niewiarowski et al., Seminars in Hematology 31(4):289, 1994). The snake venom disintegrins typically contain an RGD (Arg-Gly-Asp, SEQ ID NO:19) motif. The RGD motif is recognized by many integrins, and is present in several integrin ligands including fibronectin, vitronectin, and von Willebrand factor. Disintegrins disrupt normal adhesion processes by inhibiting the binding of cell surface integrins to their ligands.
Disintegrin-like domains have been identified in cellular proteins from both invertebrates and vertebrates (see, e.g., Westcamp and Blobel, Proc. Natl. Acad. Sci. USA 91:2748, 1994; Wolfsberg et al., Dev. Biol. 169:378, 1995; Alfandari et al., Dev. Biol. 182:314, 1997), including the ADAM family of transmembrane proteins.
B. ADAMs
The ADAMs, which have also been called MDCs, are a family of type I transmembrane cysteine-rich glycoproteins (Weskamp et al., Proc. Natl. Acad. Sci. USA, 91:2748, 1994; Wolfsberg et al., Dev. Biol. 169:378, 1995). The multidomain structure of the ADAMs typically includes an amino-terminal metalloprotease domain, a disintegrin domain, a cysteine-rich region (the region between the disintegrin domain and the transmembrane domain), a transmembrane region, and a cytoplasmic domain. At least 30 ADAM family members have been identified, in a variety of animal species. The structure of the ADAMs suggests that they may be involved in a variety of biological processes, including cell adhesion, cell fusion, signal transduction, and proteolysis. Members of the ADAM family have, in fact, been shown to play roles in sperm-egg binding and fusion, myotube formation, neurogenesis, and proteolysis.
ADAM-15, also called MDC-15 or metargidin, is the only ADAM identified to date which contains an RGD motif within its disintegrin domain. Zhang et al. (J. Biol. Chem. 273(13):7345, 1998) have reported that the isolated disintegrin domain of ADAM-15, expressed in E. coli as a glutathione S-transferase fusion protein, specifically interacts with αvβ3 integrin and that the interaction is mediated by the RGD tripeptide sequence. The recombinant fusion protein did not interact with other integrins tested, including αIIbβ3 and α5β1. Nath et al. (J. Cell Science 112:579, 1999) have reported that the entire ADAM-15 extracellular domain, expressed as an Fc fusion protein in COS cells, interacts with αvβ3 and α5β1 integrins on hematopoietic cells and that the interaction is mediated by the RGD tripeptide sequence. Zhang et al. and Nath et al. commented that the RGD-dependent interaction between ADAM-15 and αvβ3 integrin suggests a role in processes such as malignancy and angiogenesis.
C. Angiogenesis
Angiogenesis, the generation of new blood vessels, is a spatially and temporally regulated process in which endothelial and smooth muscle cells proliferate, migrate, and assemble into tubes, in response to endogenous positive and negative regulatory molecules. Angiogenesis plays important roles in both normal and pathological physiology.
Under normal physiological conditions, angiogenesis is involved in fetal and embryonic development, wound healing, organ regeneration, and female reproductive remodeling processes including formation of the endometrium, corpus luteum, and placenta. Angiogenesis is stringently regulated under normal conditions, especially in adult animals, and perturbation of the regulatory controls can lead to pathological angiogenesis.
Pathological angiogenesis has been implicated in the manifestation and/or progression of inflammatory diseases, certain eye disorders, and cancer. In particular, several lines of evidence support the concept that angiogenesis is essential for the growth and persistence of solid tumors and their metastases (see, e.g., Folkman, N. Engl. J. Med. 285:1182, 1971; Folkman et al., Nature 339:58, 1989; Kim et al., Nature 362:841, 1993; Hori et al., Cancer Res., 51:6180, 1991; Zetter, Annu. Rev. Med. 49:407, 1998). The formation of new blood vessels provides a growing tumor with oxygen, nutrients, waste removal, and a conduit by which invasive cells can enter the circulatory system and establish distant metastases. Various classes of angiogenesis inhibitors are presently being developed and tested for the prevention (e.g., treatment of premalignant conditions), intervention (e.g., treatment of small tumors), and regression (e.g., treatment of large tumors) of cancers (see, e.g., Bergers et al., Science 284:808, 1999) and other forms of pathological angiogenesis. Because many steps in the angiogenic process, including endothelial cell migration, proliferation, and morphogenesis require vascular cell adhesion, certain integrin antagonists have been tested as anti-angiogenic agents.
Several integrins are expressed on the surface of cultured endothelial and smooth muscle cells, including αvβ3 integrin. The αvβ3 integrin is an endothelial cell receptor for von Willebrand factor, fibrin, fibrinogen, and fibronectin, and a marker of angiogenic vascular tissue. Brooks et al. have reported that monoclonal antibodies to αvβ3 integrin, as well as cyclic peptide inhibitors, disrupt angiogenesis and that αvβ3 antibodies promote tumor regression (Science 264:569, 1994; Cell 79:1157, 1994). These results suggest that αvβ3 integrin is a useful therapeutic target for diseases characterized by pathological angiogenesis.
There is great need for additional compositions and methods of antagonizing the interaction between integrins and their ligands. In particular, there is great need for additional compositions and methods of inhibiting angiogenesis for the prevention, abrogation, and mitigation of disease processes that are dependent upon pathological angiogenesis.